AICSDP-15:ACTB-mEGFP
(Plasmid #87425)

• Purpose: Homology arms and linker-mEGFP sequence for N-terminus tagging of human ACTB
• Depositing Lab: Allen Institute for Cell Science
• Publication: Roberts et al Mol Biol Cell. 2017 Oct 15;28(21):2854-2874. doi: 10.1091/mbc.E17-03-0209. Epub 2017 Aug 16.

Backbone

• Vector backbone: pUC57
• Vector type: Mammalian Expression, CRISPR ; Donor Template

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: ACTB Homology Arms with mEGFP-linker
• Alt name: ACTB
• Species: H. sapiens (human)
• Insert Size (bp): 2729
• Mutation: homology arms contain point mutations to disrupt crRNA binding sites used and point mutations specific to WTC genome
• Entrez Gene: ACTB (a.k.a. BKRNS, BNS, BRWS1, CSMH, DDS1, PS1TP5BP1, THC8)
• Tag / Fusion Protein:
  • mEGFP-linker (N terminal on insert)

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: M13-F20
• 3′ sequencing primer: M13-Rev

Resource Information

• Supplemental Documents:
  • Annotated genbank file
• Articles Citing this Plasmid:
  • 6 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Industry MTA

Depositor Comments

This plasmid has been used with locus-specific CRISPR/Cas9 to add a mEGFP tag to the N-terminus of human ACTB in WTC human induced pluripotent stem cells by the Allen Institute for Cell Science. Linker (AA) sequence: AGSGT. After protein tagging using this donor template plasmid and CRISPR/Cas9 reagents, transfected cells may exhibit varying intensity levels of fluorescence, likely due to editing precision. To obtain cells of uniform intensity levels, see our protocol for fluorescence-assisted cell sorting and subcloning of transfected cells (https://www.allencell.org/instructional-videos-and-tutorials-for-cell-methods.html) Further, we recommend PCR-based assays for identifying precisely edited clones as previously described (https://www.molbiolcell.org/doi/abs/10.1091/mbc.e17-03-0209) For more information on the entire plasmid collection, please see https://www.addgene.org/allen-institute-cell-science/ .

Please visit https://www.biorxiv.org/content/early/2017/03/31/123042 for bioRxiv preprint.